830

Bulgarian Journal of Agricultural Science, 20 (No 4) 2014, 830-833

Agricultural Academy

INNOVATIVE TECHNOLOGIES FOR CREATION OF PROBIOTIC FOODS

I. NACHEVA1, M. DONEVA1, Y. TODOROV2, P. METODIEVA1, D. MITEVA1, K. DIMOV and Tsv. TSVETKOV1
1
Institute of Cryobiology and Food Technologies, BG - 1407 Sofia, Bulgaria
2
Institute of Information and Communication Technologies, BG - 1113 Sofia, Bulgaria

Abstract
NACHEVA, I., M. DONEVA, Y. TODOROV, P. METODIEVA, D. MITEVA, K. DIMOV and Tsv. TSVETKOV,
2014. Innovative technologies for creation of probiotic foods. Bulg. J. Agric. Sci., 20: 830-833
Lyophilized probiotic products have been developed by high technology methods for nutrition prophylaxis and healthy
nutrition to ensure a maximum working capacity and well- being of each individual. In their composition are included natural
sources of essential bioelements and physiologically active substances – a complex of probiotic lactic acid bacteria, oligosac-
charides, antioxidants, vitamins and others. Their fine consistency and chemical composition create a possibility for rhythmi-
cal introduction in the organism of nutritious mixtures with adequate content of plastic substances and energy. Their proved
healthy effect makes them suitable to be included in combined nutrition diets for ensuring of better quality of life in terms of
the effective prevention and improvement of the health status of the population
Key words: probiotic foods, artificial neural networks (ANN), lyofilization, nutrition diets

Introduction
The last two decades are characterized by an expressed
orientation towards more and more mass application of natu-
ral means for protecting of human health under the form of
probiotics, functional foods, biological stimulators, regulators
and other. This imposes the dynamically growing food indus-
try to develop and implement products meeting the modern
requirements for healthy nutrition (Chomakov, 2007).
Every food attributed to the functional foods is assessed
by its nutritious value, high-calorie, taste qualities and its ef-
fects on human health, expressed in supporting of the organ-
ism and healing capacity in case of disease (Georgieva and
Tzvetkov, 2001).
Among the multitude of probiotic foods, the interest in
the production and consumption of fermented dairy products,
containing live microorganisms is particularly great. They
have a favorable effect on the intestinal microflora, stimulate
selectively its growth and metabolism, strengthening in such
a way human health (De Vuyst, 2000; Cash et al., 2005).
The probiotic effect of the lactic acid microorganisms is
motivated by a number of physiological characteristics and
requirements: the microorganisms of the microflora should
be natural inhabitants of the gastrointestinal tract; they
should not be pathogenic and toxigenic; they should be ca-
pable to survive and grow under the conditions of the diges-
tive system, i.e. to be resistant to low pH and organic acids
and to maintain stable population in the gastrointestinal tract
(Denkova and Murgov, 2008).
For the development of each new food its function and pur-
pose are determined on the basis of a preliminary elaborated
concept for the interaction mechanism between its components
and the functions of the organism (Georgieva et al., 2006).
The main requirements to all technologies for food produc-
tion and storage are to obtain products with unchanged nutri-
tious value, good taste, structure and flavor (Nacheva, 2008).
By the complex combining of the ingredients in the com-
position of the new functional foods, is a high summary phys-
iological effect is achieved as a result of their application.
The quantitative proportions between the separate ingre-
dients are determined on the basis of an innovative approach
in food science, applicable in the development of new foods –­
the use of artificial neural networks (АNN).
The neural networks also find application in modeling
of the growth of various microorganisms, directly related
to the food harmlessness, prediction of physical, chemical,
functional and sensory characteristics during their produc-
tion and storage until the purchase by the consumer (Huang
et al., 2007).

This paper was presented on the workshop: ICT for Human Health and Quality of Life in the framework of the AComIn
FP7 research grant, 15-17 May, 2013, Stara Zagora, Bulgaria
E-mail: iliana_nacheva@abv.bg
Innovative Technologies for Creation of Probiotic Foods 831
The modeling through neural networks can solve relative-
ly complex tasks of the technological production processes
management, the simulation of various events and character-
istics, which provides for the obtaining of quality and harm-
less foods.
The main objective of the investigation is the creation of
a new class probiotic lyophilized foods with formulated com-
position by applying of an artificial neural network (ANN)
and with multifunctional effect.
Materials and Methods
In the composition of the new foods were included differ-
ent sources of nutrients and physiologically active substances –
probiotic complex of active lactic acid bacteria, polysaccha-
rides, fruits, grain ingredients, honey products, antocyans,
and plant oils.
Biotechnological methods
The live microflora in the probiotic products is presented
by strains lactic acid bacteria, from the collection of ICFT
and NBIMCC: Streptococcus thermophilus 1374; Lactoba-
cillus bulgaricus 1381; Lactobacterium acidophilus 1379;
Lactobacillus casei 1014, included in a combined lactic acid
starter in proportion 1:2 (cocci: rod-shaped lactic acid bac-
teria). The cultivation of the strains Lb. bulgaricum and Str.
thermophilus was realized at temperature 430С for 5-9 h, of
Lactobacterium acidophilus at temperature 370С for 5-8 h
and Lactobacillus casei subsp. casei – 300С for 5-8 h.
Technological methods
For the aims of the long term, quality and safe storage of
the probiotic foods was used the freeze-drying method (Tz-
vetkov, 1979). It was realized in a laboratory freeze-drying
installation „Gamma 1” – of company „Martin Christ”- Ger-
many with residual pressure in the vacuum chamber in the
range of 10-1–10-2 mm Hg. The freeze-drying parameters
were programmed by applying of differential thermal analy-
sis (Differential thermal analysers STA781 DTA 675 and Dif-
ferential scanning calorimeter DSC1500 of company “Stan-
ton Redcroft”, which guarantees the determining of their op-
timum values in relation to the end product quality indices).
The data from the experiments were statistically pro-
cessed with Statistics for Windows.
Modelling
The neural model was realized as a software instrument
with flexible structure which can be easily adapted to the ob-
jectives of the investigation. The obtained model was used to
formulate the composition of the experimental samples.
Analytical methods
Physical-chemical and biological investigations – resid-
ual moisture content after lyophilization (BSS 1109-89); рН;
titrated acidity, total protein – (BSS 9374-82); content of re-
ducing sugars (incl. lactose – method of Alin), vitamin С (by
the method of Muri).
Microbiological investigation –determining of the number
of viable lactic acid bacteria – by the method of the limit di-
lutions with consequent calculations by the table of МcCrady;
recording of the lactic acid microflora survival - in % - by the
following formula: % survived cells = (number of survived
cells х /number of cells 0) х100, where: х – moment of the
survival recording (at the 48th hour after the lyophilization of
the samples) 0 – moment before the lyophilization (number of
viable cells before lyophilization).
Granulating, packing and storage conditions: After the
lyophilization, the freeze-dried samples were granulated
with a granulator ”ERWEKA”, packed in a three-layer /alu-
minum - polyamide - polyethylene/ folio, hermetically vacuum
sealed and stored in premises with relative air humidity not
above 35 % at temperature - 20-220С.
Results and Discussion
The bioformulas of the new foods are in accordance with
the requirements for physiological activity, harmlessness,
safety and microbial stability valid in principle for the probi-
otic foods. Of great importance for their good assimilation is
also their taste acceptability, resp. their organoleptic proper-
ties /taste, aroma, stability of the consistency, good solubility/
which is taken in consideration in the process of formulation.
An organoleptic assessment was made, after rehydratation,
by the 9-grade Hedonic scale.
Despite the not typical taste-aroma properties due to the
specificity of their recipe compositions, the new foods are
with an excellent organolepsy and with a pleasant sour taste.
The physical-chemical indices of the investigated samples
showed the following trends (Table 1).
As it is seen from the results, the new probiotic foods are
food concentrates with low residual moisture content – 2.46 -
3.00%, which is a proof for an optimally realized technological
process of freeze-drying. The titrated acidity of the control,
before and after lyophilization is in the standard norms (up to
120оТ). The colloid character of the remaining variant samples,
causing a slight slow up of the lactic acid bacteria life intensity,
determines the decreased titrated acidity values (72 - 900Т). In
respect to the active acidity (рН), sudden changes in the values
of the investigated samples are not observed – they are in the
limits– 4.02 – 5.12, i.e. in the standard norms for fermented
foods. It is pH of the medium that also affects to a great extent
832 I. Nacheva, M. Doneva, Y. Todorov, P. Metodieva, D. Miteva, K. Dimov and Tsv. Tsvetkov

the enzyme and the biochemical activity of the microorgan-
isms, which is of importance for their viability and survival
and for their probiotic effect, respectively.
The increased content of total protein, reducing sugars
and vitamin C is a result of the introduced additional nutri-
ents in the new foods formulas.
The quantitative content and the viability of the lactic acid
bacteria are the main criteria determining the biological ac-
tivity and probiotic qualities of the experimental samples.
As it is seen from Figure 1 the survival is lowest after
lyophilization of the control without included cryoprotector
50.2% for the lactic bacteria and 68.5% for Str.thermophilus.
For the lactic acid microorganisms in the variant samples the
survival is higher – 85.4% for Str.thermophilus и 78.6% for
the lactic bacteria because of the presence in their composi-
tion of oligo- and polysaccharides, which have a cryoprotec-
tive effect as well. They increase the colloid linked water for
the sake of the osmotic pressure as a result of the filling of the
micro- and macrocapillaries (Figure 1).
For the control and the variant samples, after lyophilization
was observed was observed high resistance of Str.thermophilus
to freeze-drying – from 68.5 up to 85.4%, compared to Lb.
bulgaricus, L. acidophilus and L. casei, which are more thermo-
labile and with a lower level of survival from 50.2 up to 78.6%.
A possible reason for the different resistance of the micro-
organisms to drying is the different sensibility and the cell
wall to the strains arising during freezing and drying or the
different capacity f the cell enzymes for denaturation.
The survival of the pathogenic microflora during lyophiliza-
tion is affected by the moisture content of the product, the tem-
perature parameters and the vacuum values during storage.
The carried out by the standard methods investigations of
the microbiological characteristics of the lyophilized foods
proved an absence of microorgnism seeding and of patho-
genic microflora (Table 2).
Therefore, the applied storage conditions of the studied
lyophilized probiotics - 20-220С and relative humidity up to
35% are favorable for their long storage.

Table 1
Physical-chemical indices of the experimental samples – in native status and after lyophilization
Control Variant 1 Variant 2 Variant 3
philiza-

philiza-

philiza-

philiza-

philiza-

philiza-

philiza-

philiza-
Indices
before

before

before

before
after

after

after

after
lyo-

lyo-

lyo-

lyo-

lyo-

lyo-

lyo-

lyo-
tion

tion

tion

tion

tion

tion

tion

tion
Total moisture % 87,90±0.01 2,98±0.00 83,1±0.00 2,46±0.01 84,88±0.07 3,00±0.05 82,8±0.01 2,95±0.06
Titrated acidity °Т 110 ±0.00 95 ±0.01 82±0.01 78±0.2 79±0.012 72±0.11 90±0.011 83±0.014
рН 4,20±0.03 4,16±0.04 5,12±0.03 4,32±0.03 4,43±0.11 4,02±0.00 4,82±0.09 4,11±0.01
Total protein % 4.12±0.01 16.21±0.01 14,05±0.23 29,05±0.01 16,78±0.01 35,01±0.01 18,96±0.00 38,11±0.02
Reducing sugars % 5,16±0.02 8,97±0.02 7,08±0.01 8,58±0.06 6,12±0.00 7,00±0.02 8,09±0.01 9,39±0.03
(incl. lactose)
Vit.С mg % 1,206±0.01 2,542±0.02 3,123±0.00 3,670±0.01 2,185±0.00 3,225±0.01 2,312±0.15 3,412±0.01

Table 2
Values of the microbiological seeding of the experimental samples in CFU/g and log - in native status and after lyophilization
  Total number mezophylic  
Sample type microorganisms Moulds Yeasts Coliforms
   
 I . Native  CFU/g log CFU/g log CFU/g log CFU/g log
Control 2,8.101 1.45 2.8 0.45 0,9.101 0.95 ND -
Variant 1 2,3.10 3
3.36 1,8.101
1.25 1,3.101
1.11 ND -
Variant 2 1,45.103 3.16 1,6.101 1.2 1,8.101 1.26 ND -
Variant 3 2,7.103 3.43 1,4.101 1.15 1,7.101 1.23 ND -
II. Lyophilized                  
Control 1,2.101 1.08 1.5 1.18 4.3 0.63 ND -
Variant 1 1,9.102
2.28 1,2.101
1.08 0,7.101
0.85 ND -
Variant 2 1,38.102 2.14 1,1.101 1.04 1,1.101 1.04 ND -
Variant 3 2,6.102 2.41 0.8 0.9 1,2.101 1.08 ND -
ND – Not determined
Innovative Technologies for Creation of Probiotic Foods 833
90
78.7 80.2
80 72.4
68.5 68.4
70
Survivability, %
60
50.2
50
40
30
20
10
0
Control Variant 1 Variant 2
Streptococcus thermophilus Lactobacillus species
Fig. 1.
EFFECT OF THE WATER ACTIVITY ( a w ) – The water
activity was introduced as an important controlling and vari-
able quantity in the technology of foods with long shelf life.
For the microorganisms it was established that their growth is
in correlation with аw under certain conditions.
The results obtained from the investigation of the lactic
acid bacteria survival in lyophilized probiotic foods prove
that the water activity in them s optimum, i.e. relatively low
- 0.28 -0.47, which can be explained both with the appropri-
ate physical-chemical and biochemical characteristics of the
products and by the contained therein cryoprotective water
soluble biopolymer matrices, and also as a result of the car-
ried out cryogenic cryogenic treatment of the source material
– fast freezing and freeze-drying. Probably the lactic acid
bacteria adapt themselves to low аw easily under the effect of
the mentioned factors. This can be attributed to the possible
direct effect of the added substances on the microorganisms.
A proof for the low water activity values in our probiotic
foods is the resistance to the applied heat treatment – freezing
and freeze-drying, determined by the achieved high survival
of the lactic acid bacterial microflora during freezing and lyo-
philization.
Conclusions
A complete technology for production of a new range
probiotic products based on freeze-drying, by applying of a
modern, innovative method of formulation of their composi-
tion – neural networks (ANN) has been developed.
The applied technology ensures the obtaining of multi-
component food concentrates with maximum preservation of
Received December, 2, 2013; accepted for printing February, 2, 2014.
85.4 their nutritious value and bioactive complex, in combination
78.6 with a viable and active useful lactic acid microflora.
The new lyophilized probiotic foods, technologically pro-
cessed without introducing of chemical reagents in the bio-
system are 100% organic foods with a wide application spec-
trum in the bioprophylaxis of a number of modern diseases –
gastro-intestinal, metabolic, professional and other as well as
for increasing of the adaptability of the organism to unfavor-
able environment conditions.
Variant 3
Acknowledgments
The research work reported in the paper is partly support-
ed by the project AComIn “Advanced Computing for Innova-
tion”, grant 316087, funded by the FP7 Capacity Programme
(Research Potential of Convergence Regions).
References
Chomakov, Hr., 2007. Probiotics – past, present and future,
Es.print, pages109-170
Denkova Z. and I. Murgov, 2008. Application of probiotics in the
production of foods and beverages. Bulgarian scientific society
of nutrition and dietetics – Collection “The nutrition science
before new opportunities and challenges” – Scientific reports
from the VIII National Congress on nutrition with international
participation, pp. 228 -231.
De Vuyst, L., 2000. Technology aspects related to the application
of functional starter cultures. Food Technol.Biotechnol., 38
(2): 105-112.
Georgieva, L., I. Nacheva and Tsv. Tsvetkov, 2006.Contempo-
rary cryobiotechnological approach for safe preservation of pro-
biotic products, Abstr.book – 3-rd Central European Congress
on Food, Sofia, p. 134.
Georgieva, L. and Tzv. Tzvetkov, 2001. Scientific applied aspects
of lyophilization and its place in modern food industry. Food
Industry, 5: 3-4.
Huang, Y., L. Kangas and B. Rasco, 2007. Applications of arti-
ficial neural networks (ANNs) in food science. Crit. Rev Food
Sci. Nutr., 47 (2): 113-26.
Nacheva, I., 2008. Place and role of the buffalo’s milk in the
nutritional regime of the bulgarians. Part II. New type of
synbiotic foods, containing buffalo milk. Journal of Food Pro-
cessing Industry, 3: 41- 44.
Sean, B. Cash, Cindy Wang and Ellen W., 2005. Dairy Products
and Consumer Demand for Health Foods. Goddard Advances in
Dairy Technology, 17: 67.
Tzvetkov, Tzv., 1979. Cryobiology and Lyophilization. С., Zemiz-
dat, 134 pp. (Bg).